USE OF CHARGED HAPTENS IN CONJUGATES E.G. FOR IMPROVING REGENERATION OF SENSOR SURFACES

Abstract: In an assay method for detecting an analyte in a sample, which method comprises the step of detecting the binding of antibody to a conjugate between one or more haptens and a conjugate partner, each hapten residue of the conjugate carries a net charge. A reagent kit comprises (i) either such a hapten conjugate or a hapten capable of carrying a net charge, and (ii) an antibody directed to the hapten.

Description: USE OF CHARGED HAPTENS IN CONJUGATES EG. FOR IMPROVING REGENERATION OF SENSOR SURFACES The present invention relates to an improvement in i munoassays, and more particularly in immunoassays of the type which comprises the step of binding antibody to a hapten conjugate, which may be directly or indirectly bound to a solid phase. Antibodies are immunoglobulin molecules (serum proteins) of which there are several classes. Each class has its own characteristic molecular size, electrophoretic migration velocity, carbohydrate content, etc. Antibodies can be broken into fragments, such as the Fab fragment and the Fc fragment. The Fab fragment, like intact antibodies, can bind antigenic substances, e.g. antigens and haptens. An antigen is any substance that will provoke an immune response. In general, antigens are considered greater than 10,000 in molecular weight and are proteins, carbohydrates or glycoproteins. Smaller, less rigid molecules are not normally antigenic in pure form, but can be made so by linking them to larger molecules. These smaller molecules are called haptens. Immunoassays are generally based upon the direct or indirect detection of the formarion of a complex between the analyte and an analyte-specific antibody. Conventional immunoassays mainly belong to one of the sandwich assay format, the displacement assay format and the competition assay format. In a sandwich assay, the analyte is bi- or polyfunctional and forms a complex with an immobilized first analyte-specific antibody and a second analyte- specific antibody. In a. competition assay, the analyte competes with an analyte analogue for the binding to an analyte-specific antibody, and in a displacement assay, the analyte displaces an analyte analogue from a complex thereof with an analyte-specific antibody. In solid phase assays of these formats, the analyte complex is bound to a solid phase surface. The detection of the analyte complex formation may be performed via detectable labels, such as a radiolabel, a fluorophore or a substrate-specific enzyme, on the analyte complex or the analyte analogue complex, depending on the assay format. Alternatively, the detection may be based upon a mass-sensing principle, such as in reflection- optical methods, where the change in optical density at a solid phase surface is measured as indicative of the antibody interactions at the surface. In order to increase the specificity of the above- mentioned immunoassay formats, the binding of an additional antibody reagent, which binds to either the secondary antibody in a sandwich assay, or the analyte or analyte analogue in a competition or displacement assay, is detected. Simultaneously the sensitivity may be increased or amplified by providing more than one binding site for the additional antibody reagent on its antibody target, for example by conjugating several haptens thereto, such that several of these additional antibody reagents will bind thereto and thereby increase the response. In a sandwich assay, for instance, the secondary antibody carries two or more haptens, to which a tertiary antibody reagent is directed. Such an assay system is disclosed in inter alia US-A-4,271,140. In rechargeable, or regeneratable, immunoassay systems, the solid phase surface supports an immobilized analyte-specific antibody, from which the analyte or an analyte analogue is eluted after the assay is completed to prepare the analyte-specific surface for a subsequent assay. For efficient elution, and particularly in the case of using hapten-conjugated antibodies, it is necessary that not only the analyte and, when relevant, the analyte analogue, dissociate from the immobilized antibody, but also that any hapten-specific antibody reagents bound to the haptens in the assay dissociate from the hapten- conjugated antibody. Rechargeable receptor surfaces are used in automated assay systems, usually of the flow cell type. The present invention relates to an improvement in assays of the above-mentioned type which include the use of hapten-conjugated tibodies. In accordance with the present inventive icept this improvement consists in designing each hapten in the conjugate such that it carries a net charge. The provision of such a net charge, which advantageously may be pH-dependent, on the hapten or haptens offers inter alia the following advantages. Firstly, it makes it possible to preserve the original charge distribution of the antibody conjugated to the hapten and thereby the original physical properties of the antibody. Further advantages of such charge preservation will be explained in more detail below. Furthermore, the use of a charged hapten makes it possible to maintain the water solubility of the conjugate, which would otherwise tend to precipitate. Finally, the provision of a charged hapten will facilitate the dissociation of antibody from a complex of the antibody with the hapten conjugate, such as at low pH- values, which as indicated above is of importance for regeneratable solid assay surfaces. In one aspect the present invention therefore provides a solid phase assay method for detecting an analyte in a sample, comprising the step of detecting the binding of antibody to a conjugate between one or more haptens and a conjugate partner, which method is characterized in that each hapten residue of the conjugate carries a net charge. In another aspect the ir,.^ntion provides a reagent kit for use in a solid phase assay, which kit comprises (i) a hapten conjugate which has one or more haptens, each hapten carrying a net charge, and (ii) an antibody directed to the hapten. In still another aspect the invention provides a reagent kit for use in a solid phase assay, which kit comprises (i) a hapten which carries or is capable of carrying a net charge, and (ii) an antibody directed to the hapten. As used herein the term antibody means the intact immunoglobulin as well as any active fragments thereof, including recombinantly produced antibodies and antibody fragments. In the hapten conjugate mentioned above, the conjugate partner to which the hapten or haptens are conjugated is usually an antibody, preferably a monoclonal antibody, but also other molecules having the specific binding properties required by a particular assay format may be used. The haptens may be bound to the antibody directly or indirectly via a linking structure, such as a polymer chain. When directly bound, the number of haptens in the conjugate is preferably at least two, and preferably at least 10, more preferably at least 20. The upper limit is, of course, determined by the number of modifiable groups on the binding antibody and the effect of such modification on the biological activity of the antibody. When the haptens are indirectly bound via a linking structure, the number of haptens in each linking structure is preferably at least 10, and more preferably at least 20. The above indicated advantages of the invention, i.e. that each hapten contains a (preferably pH-dependent) net charge, will now be explained in more detail. Normally, when preparing a hapten-antibody conjugate, a hapten is coupled to the antibody, such as the secondary antibody in a sandwich assay, via a group on the antibody that is charged at neutral and acidic buffer conditions. Through this coupling, the charge on the coupling group is usually (depending on the coupling reagent used) eliminated. However, by designing the hapten in accordance with the invention such that it contains a charge equal to that of the charged group, the original charge of the coupling group is substantially preserved. Thereby the original charge distribution of the antibody is preserved, i.e. the isoelectric point, I p , also called isoionic point, is virtually unchanged. For example, in the case of the hapten being coupled to lysine residues of the antibody, which groups are positively charged in neutral and acid buffers, the hapten should contain a positive charge, e.g. an amino group. Such preservation of the antibody I p makes it possible to take advantage of this charge to electrostatically concentrate the antibody-hapten conjugate to a solid phase surface (e.g. the immobilized first antibody-analyte complex in a sandwich assay) which has an opposite charge at a selected pH and low ionic strength, as is described for unconjugated antibodies in WO 92/06380 (the disclosure of which is incorporated by reference herein) . In contrast, when, for example, a neutral hapten is conjugated to lysine residues of the antibody, the antibody conjugate will normally (depending on the coupling reagent used) obtain a lower I p value than that of the unmodified antibody, since the number of amino groups decreases. This is especially pronounced for high modification degrees, which are normally necessary to obtain the desired enhancement in the binding to the hapten-antibody conjugate. Since neutral haptens may have a limited water solubility, a conjugate of the hapten with an antibody may tend to precipitate, especially when several haptens are conjugated to the antibody. The use of charged haptens in accordance with the invention will, however, not have such a negative influence on the solubility of the conjugate and permit the solubility of the original antibody to be maintained. As mentioned above, the presence of a charged hapten also favourably affects the dissociation of an antibody, which has been bound to the hapten conjugate in the assay, at low pH values (pH 1.5 - 3.0). Thus, consider a solid phase surface having a receptor, such as antibody, immobilized thereon. For the receptor-coated surface to be efficiently rechargeable, or regeneratable, after, for instance, a sandwich assay cycle using a secondary reagent in the form of a hapten-antibody conjugate and a tertiary antibody reagent capable of binding to the hapten, it is necessary that not only the analyte and the secondary reagent be dissociated from the immobilized receptor, but also that tertiary antibody bound to the hapten conjugate be dissociated from the hapten under these conditions in order to provide for effective dissolution of the complexes formed. Low pH values are, however, usually not effective for sufficiently affecting the binding between a normal, i.e. neutral, hapten and an antibody to accomplish dissociation. In contrast, by providing a charged hapten according to the invention a dissociatable hapten-antibody system may be produced. Without ascribing this phenomenon to any special theory, it is assumed that when the hapten contains a charged group, the interaction forces between the hapten and its antibody will to a significant part consist of electrostatic forces which may be at least partially neutralized by lowering the pH. For example, if the hapten has a positive charge, the binding part of antibodies directed against the hapten would, in neutral buffers, contain a negatively charged group, i.e. a carboxylate anion (-COO-) . It would be possible to prepare high affinity antibodies of this kind by monoclonal antibody techniques. These antibody-hapten systems would be regeneratable in low pH environments where the carboxylate anion is protonated to acid form (-COOH) , as the electrostatic interaction thereby ceases and the total affinity then decreases to such an extent that a rapid dissociation is obtained. The pH value where this process takes place will vary from system to system, depending on the individual pK a value for the carboxylic acid (normally about pH 4) . The corresponding reasoning could be followed for a negatively charged hapten which is bound to an antibody having a positive charge at the binding part thereof. It is readily understood that in this case the hapten should couple to negative groups on the secondary antibody. In analogy with the above, systems are also conceivable where basic regeneration is used. In these cases the net charge of the hapten should be pH-dependent. The hapten may e.g. have a positive charge in a neutral environment, which charge is neutralized at pH values of about 9-10 and above. Examples of groups having such properties are primary, secondary and tertiary amines (but not quaternary amines) . Also negatively charged haptens may, however, be used. In that case the affinity of the positively charged binding part of the antibody is instead neutralized when the pH is increased. While the dissociation of antibody from a complex thereof with the charged hapten conjugate has been described above with regard to a pH-change, it is also possible to reduce the electrostatic interaction in the complex by changing the salt concentration of the surrounding medium, optionally in combination with a pH change. The hapten conjugate is, as mentioned above, usually a conjugate of the hapten with an antibody. The hapten or haptens may then be coupled directly to the antibody by a covalent bond. Alternatively, the hapten or haptens are indirectly coupled to the antibody via a linking structure, usually a polymer chain, i.e. the haptens are coupled to a polymer which is in turn coupled to the antibody. In this way further advantages may be obtained: (i) a greater number of haptens per antibody may be achieved and thereby a higher degree of amplification; and (ii) a lower degree of modification of the antibody is obtained, which provides for better chances that the antibody will retain its antigen-binding properties. The latter technique has previously been used in e.g. tumour therapy to provide antibodies directed against tumour cells with a relatively large number of molecules that are toxic to the cell. For the present purposes, however, the polymer is to be selected such that an optimal charge distribution is obtained for the conjugate. Thus, for a positively charged hapten the polymer should contain negatively charged groups in such a way that the introduced positive hapten charge is balanced by negative charges of the polymer chain. Alternatively, the positively charged hapten itself may be modified to also contain a negative charge to provide a zwitter-ionic hapten derivative that is then coupled to a neutral polymer such that the zwitter- ionic structure is retained. This conjugate is then coupled to the antibody. The size of the polymer may be varied from relatively small to large molecules. The lower size limit is set by the possibility of coupling a sufficient amount of haptens to obtain the desired degree of amplification, and would in practice be about Mw 10,000. The upper size limit of the polymer conjugate is determined by the considerably impaired diffusion and availability properties resulting from too large a molecule and would in practice amount to about Mw 100,000 - 200,000. The number of polymers per antibody may be from one to several, some of the advantages of this coupling technique, such as antigen-binding capability, however, being lost at higher conjugation degrees. The number of haptens per polymer chain will, of course, depend on the size of the chain. It may, however, be at least 10, and preferably at least 20, e.g. about 50. Suitable assay formats for making use of the present invention are readily apparent to the skilled person. Exemplary are, as already mentioned previously, competition assays, displacement assays and sandwich assays. Likewise, various detection principles known in the art may be used to take advantage of the invention. A preferred type of detection principle comprises detecting a measurable change in a property of a sensing structure caused by interaction of the hapten conjugate at the sensing structure surface. Among these methods are, for example, mass detecting methods, such as piezoelectric, optical, thermo-optical and surface acoustic wave (SAW) methods, and electrochemical methods, such as potentiometric, conductometric, amperometric and capacitance methods. Among optical methods may particularly be mentioned those that detect mass surface concentration, such as reflection-optical methods, including both internal and external reflection methods, e.g. ellipsometry and evanescent wave spectroscopy (EWS) , the latter including surface plasmon resonance spectroscopy (SPRS) , Brewster angle refractometry, critical angle refractometry, frustrated total reflection (FTR) , evanescent wave ellipsometry, scattered total internal reflection (STIR) , optical wave guide sensors, evanescent wave based imaging, such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, etc. , as well as methods based on evanescent fluorescence (TIRF) and phosphorescence. Among the optical methods mentioned above, especially SPR has attracted much attention recently. The phenomenon of SPR is well known. In brief, SPR is observed as a dip in intensity of light reflected at a specific angle from the interface between an optically transparent material, e.g. glass, and a thin metal film, usually silver or gold, and depends on among other factors the refractive index of the medium (e.g. a sample solution) close to the metal surface. A change of refractive index at the metal surface, such as by the adsorption or binding of material thereto, will cause a corresponding shift in the angle at which SPR occurs. To couple the light to the interface such that SPR arises, two alternative arrangements are used, either a metallized diffraction grating (Wood's effect) , or a metallized glass prism or a prism in optical contact with a metallized glass substrate (Kretschmann effect) . For further details on SPR, reference is made to our WO 90/05295. In an SPR-based immunoassay, a ligand may be bound to the metal surface, and the interaction thereof with an analyte of an aqueous sample in contact with the surface is monitored. As already mentioned above, a reagent kit for performing the method of the invention may comprise a hapten conjugate and an antibody capable of binding to the hapten. Alternatively, conjugation of the hapten may be performed by the user, and in such a case the kit will comprise instead of the hapten conjugate, a hapten, which optionally is preactivated for the conjugation, and which carries or by simple means may be made to carry a net charge. In the following Examples, which are intended to illustrate the present invention, by way of example only, a commercial biosensor system based on the Kretschmann effect (briefly mentioned above) was used (BIAcore™, supplied by Pharmacia Biosensor AB, Uppsala, Sweden) . Reference will be made to the accompanying drawings, wherein: Fig. 1 is a sensorgra where the response in Resonance Units (RU) is plotted against time (in seconds) for the assay procedure in Example 1; Fig. 2 is a corresponding sensorgram to that in Fig. 1 for the assay procedure in Example 2, and Fig. 3 is a sensorgram where the response in Resonance Units (RU) is plotted against time (in seconds) for the assay procedure in Example 3. EXAMPLE 1 Hapten synthesis and antibody preparation The N-hydroxysuccinimide ester of the hapten N-(p- nitrobenzyl)-N-methyl-aminovaleric acid was synthesized as described by Shokat et al., Nature 338 (1989) 269. Accordingly, this ester was reacted with the carrier protein keyhole limpet haemocyanin to produce an immunoconjugate which was used for immunization of mice in order to generate monoclonal antibodies directed against the hapten structure. Immunization, fusions and screening were carried out by using standard methods. From several antibody candidates, one monoclonal antibody was selected to be used as tertiary antibody in an assay for luteinizing hormone (LH) . Preparation of antibody-hapten conjugates Antibody conjugates with the hapten derivative described above were prepared by the addition of 80 μl of a 40 mM solution of the hapten derivative in water to 120 μl of a solution of a monoclonal antibody (anti-LH, 6.7 mg/ml) in 16 mM sodium phosphate buffer with 0.25 M sodium chloride, pH 8.5. The reaction mixture was incubated at 4°C for 4 hours, diluted with 800 μl of 0.15 M sodium chloride and dialyzed in 0.15 M sodium chloride at 4°C for 18 hours. Assay for luteinizing hormone (LH) Measurement of LH was carried out in a BIAcore™ instrument (Pharmacia Biosensor AB, Uppsala, Sweden) with an anti-LH monoclonal antibody (clone 569, Pharmacia AB, Uppsala, Sweden) immobilized to a CM5 Sensor chip (Pharmacia Biosensor AB, Uppsala, Sweden) using standard amine coupling chemistry (Johnsson et al., Analytical Biochemistry, 198 (1991) 268-277) . Figure 1 is a so-called sensorgram showing the response signals as a function of time for (1) the binding of anti-hapten antibody as zero- control (50 μg/ml in 10 mM acetate pH 5.0), (2) regeneration (10 mM glycine-HCl, pH 2.0), (3) LH (100 ng/ml in running buffer) , (4) anti-LH conjugate (50 μg/ml in 10 mM acetate) , (5) the anti-hapten antibody, (6) regeneration (as in 2 above) and (7) after regeneration. Events (1) to (6) take place shortly (about 30 seconds) after each measurement point indicated by "X" and the corresponding figure in the sensorgram, whereas (7) represents the measurement at the respective "X". A running buffer composed of 10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA and 0.05% Tween 20, pH 7.4, was used in the assay. The absolute and relative responses (the responses relative to the preceding measurement value) at the measurement times indicated by 11 X" in the sensorgram in Fig. 1 are listed in Table 1 below. TABLE 1 From Table 1 it is seen that the effect of the anti- hapten antibody enhances the LH response by about 35 times (1745-67)/49) , including a correction for the zero-control. Compared to the ordinary assay format, the additional anti- hapten antibody step enhances the secondary antibody signal by 7.9 times. As both antibodies are of the IgG type with a molecular weight of approximately 150.000 D, this indicates that each secondary antibody binds overall 7.9 anti-hapten antibodies. The sensorgram and Table 1 also show the regeneration effect of the pH 2.0 pulse where the baseline level returns to the same level as before the assay cycle, indicating a complete removal of all biospecifically bound material and thus the reversibility of the anti-hapten binding. EXAMPLE 2 (COMPARATIVE) Preparation of antibody-hapten conjugate A reactive theophylline derivative was prepared by activation of 3-(4'-carboxybutyl)-l-methylxanthine (Hodgkinson et al., Ann. Clin. Biochem., 22 (1985) 519-525) with isobutylchloroformate (1 mole eq.) in tetrahydrofuran in the presence of triethylamine (1 mole eq.). The xanthine was partly dissolved in tetrahydrofuran, cooled on an ice bath, whereupon triethylamine was added, followed by isobutylchloroformate. The mixture was stirred on an ice bath for 1.5 hours. The tetrahydrofuran solvent of a 15 μl aliquot of the mixture was evaporated with a gentle nitrogen gas stream. A 1.5 mg/ml solution (200 μl) of anti- LH (clone 79, Pharmacia AB, Uppsala, Sweden,) was added to the residue and the reaction mixture was incubated on an ice bath for 4 hours. Low molecular weight components were separated from the antibody conjugate by gel filtration on an NAP-5 column (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) using a buffer consisting of 10 mM HEPES with 0.15 M NaCl, pH 7.4. Assay for luteinizing hormone (LH) Measurement of LH was carried out in a BIAcore™ instrument (Pharmacia Biosensor AB, Uppsala, Sweden) with an anti-LH monoclonal antibody (clone 636, Pharmacia AB, Uppsala, Sweden) immobilized to a CM5 Sensor chip (Pharmacia Biosensor AB, Uppsala, Sweden) using standard amine coupling chemistry (Johnsson et al., Analytical Biochemistry, 198 (1991) 268-277) . Figure 2 is a similar sensorgram as in Fig. 1 showing the response signals as a function of time for (1) the binding of LH (500 ng/ml in running buffer) , (2) theophylline-anti-LH conjugate (50 μ g/ml in HEPES buffer, pH 7.4), (3) anti-theophylline antibody (100 μg/ml in 10 mM acetate pH 5.0), (4) regeneration (10 mM glycine-HCl, pH 2.0) and (5) after regeneration. Events (1) to (4) take place shortly (about 30 seconds) after each measurement point indicated by "X" and the corresponding figure in the sensorgram, whereas (5) represents the measurement at the respective "X". A running buffer composed of 10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA and 0.05% Tween 20, pH 7.4, was used in the assay. The absolute and relative responses at the measurement times indicated by "X" in the sensorgram in Figure 2 are listed in Table 2 below. TABLE 2 From Table 2 it is seen that the effect of the anti- theophylline antibody enhances the LH response by 8.9 times (7290/820) . The sensorgram and Table 2 also show an incomplete regeneration effect of the pH 2.0 pulse with approximately 1330 RU remaining on the surface, in contrast to the successful removal of the bound material obtained in Example 1 (method of the invention) . EXAMPLE 3 Hapten synthesis and antibody preparation The hapten reagent 4-[ [5-[ (2,5-dioxo-l- pyrrolidinyl)oxy]-1,5-dioxopentyl]amino]-N,N-dimethyl-N- phenylbenzenemethanaminium bromide was synthesized according to Janda et al., J. Am. Che . Soc, 113. 5427- 5434 (1991) . Accordingly, this ester was reacted with the carrier protein keyhole limpet haemocyanin to produce an immunoconjugate which was used for immunization of mice in order to generate monoclonal antibodies directed against the hapten structure. Immunization, fusion and screening were carried out by using standard methods. From several antibody candidates, one monoclonal antibody was selected to be used as tertiary antibody in an assay for creatine kinase MB. Preparation of a hapten- extran polymer conjugate A solution of 5.0 g dextran T40 (Pharmacia Biotech AB, Uppsala, Sweden) in 15 ml water (Milli-Q quality) was mixed with a solution of 27.0 g chloroacetic acid in 9 M sodium hydroxide. The mixture was allowed to react at room temperature for 16 hours, thereafter neutralized with 18 % HC1 to pH 7.6 and purified by dialysis (dialysis tube, Mw cut off 1,000) against 4 x 3 1 water. The resulting solution was freeze-dried and analyzed by acid-base titration to yield a modification degree of 0.27 carboxymethyl groups/glucose unit. The resulting carboxymethyl (CM) dextran (50 mg) was dissolved in a solution of 0.4 M ethanediamine in water, pH adjusted to 6.5 \ th hydrochloric acid. A solution of ethyl-dimethylaminopropyl-carbodiimide (1 M) and water was added to give a final volume of 2.5 ml and the concentrations 261 mM of ethanediamine and 58 mM of carbodiimide. The reaction mixture was allowed to stand for 24 hours at 27 °C. The mixture was then purified by gel filtration (Pharmacia PD10, Pharmacia Biotech AB, Uppsala, Sweden) and freeze-dried. Analysis of the amount of primary amine groups was performed according to the method by Habeeb, Anal. Biochem., 14 . , 328-36 (1966) by reaction with trinitrobenzenesulfonic acid. The analysis showed that 51 % of the carboxymethyl groups had been converted into amine groups. A solution of amino-CM dextran (20 mg in 0.1 M borate buffer, pH 8.5) was allowed to react with the hapten reagent (50 mM in water) to give final concentrations of 10 and 20 mM of the amine and hapten, respectively. The mixture was reacted for 17 hours at 27 °C and was thereafter purified by gel filtration (Pharmacia PD10, Pharmacia Biotech AB, Uppsala, Sweden) and freeze-dried. The hapten content was analyzed spectrophotometrically which showed that approximately 90 % of the amine groups had reacted with the hapten. The hapten-amino-CM dextran conjugate (5 mg/ l in phosphate buffer, pH 7.5) was further reacted with an ethanol solution of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, Pharmacia Biotech AB, Uppsala, Sweden) (3 mol SPDP/mol polymer) . The mixture was reacted for one hour at room temperature, gel filtrated and analyzed spectrophotometrically which showed a modification degree of approximately two SPDP residues per polymer chain. The polymer conjugate (0.5 ml in phosphate buffer, pH 7.5) was reduced with 1,4-dithioerythritol (30 μl of a 0.1 M solution) for 30 minutes and thereafter gel filtrated. Preparation of haptenpolymer-antibody conjugate A monoclonal antibody directed against creatine kinase MB (CKMB) was modified with SPDP as follows. A 6 mg/ml solution of the antibody in 0.1 M phosphate buffer with 0.15 M NaCl, pH 7.5, was reacted with a 25 mM solution of SPDP in ethanol (80 nmol per mg antibody) . The mixture was reacted for one hour at room temperature and thereafter gel filtrated (Pharmacia NAP-10, Pharmacia Biotech AB, Uppsala, Sweden) and eluted with a 10 mM phosphate buffer with 3.4 mM EDTA, pH 6.5. The modification degree was determined spectrophotometrically to 5.6 SPDP residues per antibody. The SPDP modified antibody was reacted with the reduced polymer conjugate (3 mol polymer per mol SPDP residue on antibody) for 17 hours at room temperature. The product was purified by ultrafiltration (Centricon 100, Amicon Corp., MA, U.S.A.). Analysis of enhancement effect in assay for creatine kinase (CKMB) The enhancement effect by the antibody-polymer conjugate was analysed in a BIAcore™ instrument (Pharmacia Biosensor AB, Uppsala, Sweden) as follows. A monoclonal antibody directed against CKMB was immobilized to CM5 Sensor Chip (Pharmacia Biosensor AB, Uppsala, Sweden) using standard amine coupling chemistry (Johnsson et al.. Anal. Biochem., 198. 268-277 (1991). Figure 3 is a sensorgram showing the response signals as a function of time for (1) the binding of the analyte CKMB (200 ng/ml in "sample buffer") , (2) antibody polymer conjugate (50 μg/ml in running buffer) , (3) anti-hapten antibody (100 μg/ml in 10 mM acetate, pH 5.0) and (4) regeneration (10 mM glycine- HC1, pH 2.25). Events (1) to (4) take place shortly (about 30 seconds) after each measurement point indicated by "X" and the corresponding figure in the sensorgram, whereas (5) represents the measurement at the respective "X". The sample buffer was composed of 10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.05 % Tween 20, 9 % human serum albumin and 0.02 % sodium azide, pH 7.4. A running buffer composed of 10 mM HEPES, 0.5 M NaCl, 3.4 mM EDTA and 0.05 % Tween 20, pH 7.4, was used in the assay. The absolute and relative responses at the measurement times indicated by "X" in the sensorgram are listed in Table 3 below. TABLE 3 Related Time Absolute Relative event No. (sec. ) response (RU) response (RU) 1 65.9 21967 0 2 619.8 22219 252 3 935.3 22393 174 4 1251.2 26501 4108 5 1383.3 21979 -4522 From Table 3 it is seen that the response from the binding of the haptenpolymer-antibody complex to the CKMB analyte is amplified by about 23.6 times by the additional anti-hapten antibody step (4108/174) . The sensorgram and Table 3 also show the regeneration effect of the pH 2.25 pulse where the baseline level returns to the same level as before the assay cycle, indicating a complete removal of all biospecifically bound material and thus the reversibility of the formed complex.

Claims: CLAIMS 1. An assay method for detecting an analyte in a sample, comprising the step of detecting the binding of antibody to a conjugate between one or more haptens and a conjugate partner, characterized in that each hapten residue of the conjugate carries a net charge. 2. The method according to claim 1, characterized in that the hapten conjugate is capable of binding to the analyte or an analyte analogue. 3. The method according to claim 1 or 2, characterized in that said net charge is pH-dependent. 4. The method according to claim 1, 2 or 3, characterized in that said conjugate partner is an antibody. 5. The method according to any one of claims 1 to 4, characterized in that each hapten is coupled directly to the conjugate partner. 6. The method according to claim 5, characterized in that each hapten conjugate contains at least two, preferably at least 20 haptens. 7. The method according to any one of claims 1 to 4, characterized in that each hapten is coupled to the conjugate partner via a linking structure, e.g. a polymer chain. 8. The method according to claim 7, characterized in that each linking structure contains at least 10, preferably at least 20 haptens. 9. The method according to claim 7 or 8, characterized in that each linking structure comprises a group or groups having a balancing charge opposite to that of the ' npten or haptens. 10. The method according to claim 7 or 8, characterized in that each hapten is a zwitter-ionic derivative. 11. The method according to any one of claims 1 to 10, characterized in that the hapten conjugate is selected such that said binding of antibody to the conjugate comprises electrostatic interaction and that the method comprises the step of dissociating any complex formed between antibody and hapten conjugate by changing the salt concentration of the surrounding medium to reduce the electrostatic interaction in said complex. 12. The method according to any one of claims 1 to 11, characterized in that the hapten conjugate is selected such that said binding of antibody to the conjugate comprises electrostatic interaction and that the method comprises the step of dissociating any complex formed between antibody and hapten conjugate by changing the pH of the surrounding medium and thereby neutralizing said charge. 13. The method according to claim 12, characterized in that said hapten or haptens carry a positive charge and that the pH is changed to a low pH value. 14. The method according to claim 12, characterized in that said hapten or haptens carry a negative charge and that the pH is changed to a high pH value. 15. The method according to any one of claims 1 to 14, characterized in that the hapten conjugate has a charge distribution substantially identical to that of the non- conjugated conjugate partner. 16. The method according to any one of claims 1 to 15, characterized in that the hapten conjugate has a water solubility substantially identical to that of the non- conjugated conjugate partner. 17. The method according to any one of claims 1 to 16, characterized in that the detection principle of the assay method is evanescent wave sensing. 18. A reagent kit, comprising (i) a hapten conjugate as defined in any one of claims 1 to 10 and 15 to 16, and (ii) an antibody directed to the hapten. 19. A reagent kit, comprising (i) a hapten capable of carrying a net charge and optionally preactivated for conjugation to an antibody, and (ii) an antibody directed to the hapten.

Inventors: LOEFAAS STEFAN, LOEFAAS, STEFAN
Applicants: LOEFAAS STEFAN, PHARMACIA BIOSENSOR AB, LOEFAAS, STEFAN
Application year: 1994
Main classification: G01N33
IPC codes: G01N33